Health assessment of nesting loggerhead sea turtles (Caretta caretta) in one of their largest rookeries (central eastern Florida coast, USA)

Abstract Reproduction is a physiologically demanding process for sea turtles. Health indicators, including morphometric indices and blood analytes, provide insight into overall health, physiology and organ function for breeding sea turtles as a way to assess population-level effects. The Archie Carr National Wildlife Refuge (ACNWR) on Florida’s central eastern coast is critical nesting habitat for loggerhead sea turtles (Caretta caretta), but health variables from this location have not been documented. Objectives of the study were to (1) assess morphometrics and blood analyte data (including haematology, plasma biochemistry, protein electrophoresis, β-hydroxybutyrate, trace nutrients, vitamins and fatty acid profiles) from loggerheads nesting on or near the beaches of the ACNWR, (2) investigate correlations of body condition index (BCI) with blood analytes and (3) analyse temporal trends in morphometric and blood analyte data throughout the nesting season. Morphometric and/or blood analyte data are reported for 57 nesting loggerheads encountered between 2016 and 2019. Plasma copper and iron positively correlated with BCI. Mass tended to decline across nesting season, whereas BCI did not. Many blood analytes significantly increased or decreased across nesting season, reflecting the catabolic state and haemodynamic variations of nesting turtles. Twenty-three of 34 fatty acids declined across nesting season, which demonstrates the physiological demands of nesting turtles for vitellogenesis and reproductive activities, thus suggesting potential utility of fatty acids for the assessment of foraging status and phases of reproduction. The findings herein are relevant for future spatiotemporal and interspecies comparisons, investigating stressor effects and understanding the physiological demands in nesting sea turtles. This information provides comparative data for individual animals in rescue or managed care settings and for assessment of conservation strategies.


Introduction
Loggerhead sea turtles (Caretta caretta) are protected under the Convention on International Trade in Endangered Species Appendix I as well as the US Endangered Species Act and listed as vulnerable on the red list of the International Union for the Conservation of Nature (Casale & Tucker, 2017;IUCN 2024).Similar to other sea turtle species worldwide, loggerheads face various threats, including fisheries bycatch, climate change, boating interactions, illegal hunting, nesting beach loss or degradation, pollution and disease (Ataman et al., 2021;Bolten et al., 2011;Bolten et al., 2019;Donlan et al., 2010;Foley et al., 2019;Fuentes et al., 2016, Fuentes et al., 2023;Page-Karjian et al., 2020;Wallace et al., 2010).Major threats for eggs and hatchlings include pollution (including artificial lighting), beach degradation (including coastal armoring), increasing nest temperatures and predation (Bolten et al., 2011).
Adult female sea turtles experience physiological demands during their reproductive migration and nesting season.Loggerheads migrate hundreds of kilometres to their nesting beaches and lay 4-8 clutches at an inter-nesting interval of 10-19 days, with a remigration interval of 2-5 years (Hirsch, 2022;Miller, 1997;Tucker, 2010).The largest aggregations of nesting loggerheads worldwide are found on the central and southeastern Atlantic coast of Florida and on eastern Masirah Island in the Sultanate of Oman (Ataman et al., 2021;Casale & Tucker, 2017;Ceriani et al., 2017;Ceriani et al., 2019;Ceriani & Meylan, 2017;Ehrhart et al., 2014;Hirsch, 2022;Willson et al., 2020).Considered one of the most important regions for loggerhead nesting worldwide, the Archie Carr National Wildlife Refuge (ACNWR) is located along the beaches of central eastern Florida.Between 2014 and 2023, ACNWR beaches supported an annual average of 12 733 ± 2279 loggerhead nests along 21 km of protected coastline (personal communication from Dr E. Seney, University of Central Florida).The loggerhead population nesting in the ACNWR is part of the Northwest Atlantic Regional Management Unit, currently a stable population, but at risk of population decline if current threats remain unabated (Ceriani et al., 2017;Wallace et al., 2010).Loggerheads appear to have high site fidelity to nesting beaches, and although they may re-nest at a range of up to 290 km within one nesting season; however, intraannual nesting ranges are typically <10 km (Bjorndal et al., 1983;Hirsch, 2022;Tucker, 2010;Weishampel et al., 2003).This site fidelity is thought to divide Florida nesting loggerheads amongst four genetically distinct management populations, within which ACNWR turtles occupy a central eastern Florida group (Shamblin et al., 2011).
In addition to the high physiological demands of reproductive activities (e.g.migration, vitellogenesis, nesting), nesting loggerheads undergo times of hyporexia as part of a capital breeding strategy, which adds to their metabolic challenges during this phase of their life cycle (Perrault & Stacy, 2018).These characteristics highlight the importance of long-term health monitoring in sea turtles to better understand their populations and the value of health surveillance data to inform policymakers (Fuentes et al., 2023;Page-Karjian & Perrault, 2021).
To answer specific questions regarding population health, health assessment studies in wildlife species aim to collect targeted samples from live animals in a way that minimizes effects from sampling on the animals' behavioural and physiological states.Establishment of baseline health variables for loggerheads is considered a priority action in the most recent recovery plan for the Northwest Atlantic population of loggerheads (Fuentes et al., 2023;Hamann et al., 2010;Bolten et al., 2019;NMFS & USFWS, 2008).
Blood is a sample matrix that can be used to gain information on many health variables.Sampling blood is minimally invasive and causes low risk of disturbing the nesting process if performed per standardized recommendations (NMFS SEFSC, 2008).Establishment of blood analyte baselines for various sea turtle species from different geographical sites and life-stage classes allows a better understanding of population health, is important for evaluating impacts of stressors on the population and may be useful for improving rehabilitation outcomes and animal welfare measures.For these reasons, blood analyte data have been reported for many sea turtle species, life-stage classes, from various geographical areas and in different settings (Aguirre & Balazs, 2000;Fleming et al., 2020;Flint et al., 2010, Flint et al., 2015;Kelly et al., 2015;Osborne et al., 2010;Page-Karjian et al., 2015, Page-Karjian et al., 2020;Perrault et al., 2014, Perrault et al., 2020, Perrault et al., 2022;Stacy & Innis 2017;Stacy et al., 2018, Stacy et al., 2019, Stacy et al., 2023).However, blood data from nesting loggerheads at Florida's central eastern coast have not been published to date.Nesting females represent an important life stage for collecting health data given their unique physiological state during phases of active reproduction and their value in maintaining the population (Goldberg et al., 2013;Hays et al., 2002;Honarvar et al., 2011;Page-Karjian et al., 2020;Perrault et al., 2012, Perrault et al., 2014, Perrault et al., 2016;Perrault & Stacy, 2018;Plot et al., 2013;Smith, 2010).Health variables of nesting females, including morphometric data and blood analyses related to metabolism and nutrition, provide valuable insight into physiological changes associated with capital breeding during nesting seasons and for geographical variations amongst populations.The objectives of this study were to (1) assess morphometrics and comprehensive blood analyte data (including haematology, routine plasma biochemistry, protein electrophoresis, βhydroxybutyrate [BHB], trace nutrients, vitamins and fatty acid profiles) from loggerheads nesting on or near the beaches of the ACNWR, (2) investigate correlations of body condition index (BCI) with blood analytes and (3) analyse temporal trends in morphometric and blood analyte data across nesting season.

Study site
Loggerheads nesting within or near the ACNWR in Brevard and Indian River Counties, Florida, USA were sampled for this study (Fig. 1).Loggerheads nesting in Brevard County were sampled on the beach between 27.9536

Blood sampling and morphometric measurements
Trained researchers patrolled the ACNWR on foot at night, between 2100 and 0400 h, May-August 2017-19 (with one pilot trip in August 2016).Turtles were not approached until they entered the laying phase of nesting, when they are minimally responsive to external stimuli and unlikely to abandon nesting (i.e.nesting fixed action pattern) (Hailman & Elowson, 1992;Smith et al., 2021).Blood collection during oviposition also ensured safe blood draws without manual restraint, as turtles are immobile during this phase of nesting.The venipuncture site (external jugular vein) was cleaned of debris with fresh water, followed by three alternating repetitions of 0.4% chlorhexidine gluconate solution (Phoenix™, Elgin, IL) and 70% isopropyl alcohol (Aspen Veterinary Resources, LTD., Greeley, CO).Up to 30 ml of blood were collected using 18-to 21-gauge, 1.5-to 2-in needles (BD PrecisionGlide™, Becton Dickinson and Co., Franklin Lakes, NJ) attached to 6-ml syringes (Monoject™, Covi-dien™/Medtronic, Minneapolis, MN) and an extension set (Baxter International Inc., Deerfield, IL).The blood collection process lasted 2-4 min.Blood was immediately transferred into various tubes, including green top (lithium-heparin), royal blue top (sodium EDTA), red top with serum separator and red top tube (Becton Dickinson and Co.).Additionally, one drop (∼1-5 μl) of whole blood from the lithium-heparin tube was used to determine glucose using a point-of-care glucometer (Accu-Chek ® , Roche Diabetes Care, Inc., Indianapolis, IN).The blood tubes were insulated with bubble wrap in the field and transported on regular wet ice to Disney Animal Health's in-house laboratory (Disney's Animal Kingdom ® , Orlando, FL), where the samples were processed within 12 h after blood collection.
Following venipuncture, morphometric measurements were taken including standard straight carapace length (SCL), minimum straight carapace length (SCL min ), straight carapace width (SCW) and head width using Haglöf-Mantax (Haglöf Inc., Madison, MS) aluminium callipers.Minimum curved carapace length (CCL min ) and curved carapace width (CCW) were measured using a flexible measuring tape.An external physical examination was also conducted (Deem & Harris, 2017;Page-Karjian & Perrault, 2021) to include evaluation of the eyes and nose and categorization of the scute and skin condition.Subjective body condition was assessed as being either 'poor', 'fair' or 'good', based on visual assessment, including evaluation of robust or sunken-appearing eyes, and subjective observation of soft tissue thickness of shoulder and neck.The presence and/or prevalence of epibionts with the potential to affect or indicate health, including cloacal marine leeches (Ozobranchus sp.), imbedded barnacles (Platylepadidae spp.) and commensal benthic taxa (e.g.invertebrates, macroalgae) were noted during the physical examination (Stacy & Innis, 2017, Stamper et al., 2005) and classified as 'none', 'low', 'medium' or 'high'.Presence or absence of external lesions, including evidence of trauma (e.g.healed or active wounds) was recorded.
If previous tags were absent on the turtles, including scanning the front flippers for passive integrative transponder (PIT) tags with a microchip reader (Biomark GPR+, Boise, ID), Inconel tags (style 681; National Band and Tag Co., Newport, KY) were applied to the first scale proximal to the shell on the trailing edge of the turtles' front flippers.In addition, a PIT tag was inserted into the triceps muscle of the right front flipper.Prior to tagging, the application site was cleaned using a sterile alcohol wipe at least three times.
Mass of sampled turtles was obtained during their return to sea after the nest camouflaging phase was complete.From 2016-17, soft nylon mesh (5.4-cm bar) scallop netting was placed on the sand ahead of the turtle's projected route.The turtle was guided into the centre of the net, at which point she was quickly secured with the netting, which was then attached to a metal bar using a 10-cm shackle and lifted by two people.The shackle was fastened to a portable digital hanging scale (Handy Roughneck™, Unbranded, Northern Tool and Equipment, Burnsville, MN) to collect mass.From 2017-19, the metal bar was replaced with an aluminium tripod.For both methods, the turtle was elevated above the sand so that none of her extremities were touching the sand.After mass was recorded, the turtle was lowered and released.In 2019, the protocol was slightly adjusted to replace the scallop netting with a customized turtle sling.Scales were tared to the net or sling prior to weighing.BCI was calculated using the formula from Bjorndal et al. (2000): BCI = mass SCL 3 × 10, 000

Blood processing and analysis
At Disney's in-house laboratory, well-mixed whole blood from each lithium-heparin tube was used to determine packed cell volume (PCV) using 75-mm non-heparinized capillary tubes (Jorgensen Laboratories, Inc. Loveland, CO) that were centrifuged for 3 min at 15 000 rpm (27 670 g) in a microhaematocrit centrifuge (Zip-IQ, LW Scientific, Lawrenceville, GA).After centrifugation, plasma colour and transparency were visually assessed for haemolysis, lipemia or other discoloration (Stacy & Innis, 2017).Plasma total protein (TP-R) and fibrinogen (heat precipitation method) were determined by standardized methods using a digital refractometer (AR200, Reichert ® Technologies/Ametek ® , Depew, NY) using the plasma portion from the capillary tube.
Up to four blood smears (from lithium-heparin tube) were prepared followed by air drying and fixation in >99% methanol (Thermo Fisher Scientific, West Palm Beach, FL) The remaining whole blood from all tubes was centrifuged (LW Ultra Select, LW Scientific, Lawrenceville, GA) at 3500 rpm (1507 g) for 15 min and plasma or serum was aliquoted into 2-ml cryogenic vials.All plasma and serum samples were stored frozen in an ultra-low cryogenic freezer at −80 • C (Revco-EXF, Thermo Fisher Scientific, Marietta, OH) for up to 4 months prior to distribution and shipping in batches on dry ice.Analyses were run each year after completion of the nesting season at consistent diagnostic laboratories between 2017 and 2019.
Serum trace nutrient concentrations (from royal blue top tubes) including cobalt, copper, iron, manganese, molybdenum, selenium and zinc were measured using inductively coupled plasma-mass spectrometry (ICP-MS) using standards from Inorganic Ventures (Christiansburg, VA) at Michigan State University (MSU) Veterinary Diagnostic Laboratory (Lansing, MI).Serum iron concentrations were determined using an Olympus AU640e (Shinjuku City, Tokyo, Japan).β-hydroxybutyrate was quantified from lithium-heparinized plasma at MSU using the Catachem colorimetric assay (Catachem, Inc., Oxford, CT) on a Beckman Coulter AU series analyser (Brea, CA).
Serum vitamin concentrations (from royal blue top tubes) including retinol (i.e.vitamin A) and vitamin E isoforms (αtocopherol, γ-tocopherol and δ-tocopherol) were determined using high-performance liquid chromatography (HPLC) at Eurofins Craft Technologies (Wilson, NC).
Fatty acid profiles were quantified in serum (from red top tubes) at Lipid Technologies (Austin, MN).Separation, detection and quantification were accomplished by capillary gas chromatography utilizing a 30-m Restek free fatty acid phase coating (Restek Corp., Bellefonte, PA) and EZChrom software (Scientific Software International Inc., Lincolnwood, IL).

Statistical analysis
Measures of central tendency, range and 95% reference intervals with 90% confidence intervals for blood analytes are presented in standard international units for all study turtles (none had to be excluded due to injuries or abnormalities).Parametric methods for sample sizes ≥20 but <120 were used to calculate reference intervals, unless otherwise indicated (Friedrichs et al. 2012).Normality was assessed using the Shapiro-Wilk test, and outliers were detected using the Dixon-Reed test.All plasma samples included in the calculation of reference intervals had haemolysis and lipaemia scores ≤1+, which is considered to not cause interferences using dry chemistry analysis.Reference intervals could not be calculated for some variables due to low sample sizes (<20) or because most values fell below the detection limits (i.e.right-skewed data); these data are reported descriptively.Least-squares linear regressions (with data transformations as necessary) were used to compare mass and SCL; BCI and blood analytes; and total protein concentrations determined by refractometer and the Biuret method.Blood analyte data were combined by month across years and an analysis of variance (ANOVA) with Tukey's post hoc tests was used to compare these data by month.Kruskal-Wallis tests with Dunn's post hoc tests with Benjamini-Hochberg adjustment were used when the data did not meet the assumptions of normality.Kruskal-Wallis tests were also used to compare categorical data by month (e.g.subjective body condition, epibiont loads and presence of cloacal leeches).

Physical examination and morphometrics
A total of 57 nesting loggerheads were sampled between 18 August 2016 and 17 August 2019.One turtle was sampled in 2016 (on 18 August), 9 in 2017 (from 4 May to 8 August), 24 in 2018 (from 12 May to 17 August) and 23 in 2019 (from 11 May to 17 August).Eight turtles were previously tagged, 48 turtles had no tags and new tags were applied and tagging information was missing for 1 turtle.
None of the nesting females showed evidence of behavioural or major external abnormalities or lesions; minor injuries were limited to the flippers and carapace.No turtles had external abnormalities to the rhamphothecae; however, 16 of 54 (30%) turtles presented with minimal, healed injuries including small divots in the carapacial scutes, minor and/or superficial damage to the posterior carapace, minor injuries to the hind limbs or healed partial amputations to the front flippers and hind limbs (Table 1).None of the turtles showed abnormal behaviour or abandoned their nesting attempts.
All turtles were in fair to good body condition (Table 1) based on subjective observation of thickness of shoulder and neck soft tissues and BCI (Harris et al. 2017)   are reported in Table 1.Mass and SCL showed a strong linear relationship (r 2 = 0.77; P < 0.001; n = 40; Fig. 2).
Generally, turtles included in this study had low to moderate skin and carapace epibiota that consisted largely of Stomatolepas spp.barnacles and macroalgae.The majority of turtles (84%: 37 of 44) had leeches within their cloaca.Subjective BCI and prevalence of cloacal leeches, Stomatolepas spp.barnacles and macroalgae did not significantly differ by month (P > 0.050 in all cases).Epibiota load (scored as none, low, medium and high) significantly differed by month (H(3) = 13.148;P = 0.004), with loggerheads nesting in May having significantly fewer epibionts than loggerheads nesting in July (P = 0.002) and August (P = 0.016).

Reference intervals
Samples with moderate (2+) to marked (3+) haemolysis (n = 12) and moderate (2+) lipaemia (n = 1) were removed from calculation of reference intervals (Stacy & Innis 2017;Stacy et al., 2019).Lymph contamination was not observed in any blood samples during collection.Measures of central tendency, range and reference intervals in Standard International units are reported for haematology and plasma biochemistry data in Table 2; protein electrophoresis, vitamins, trace nutrients and BHB in Table 3; and fatty acids in Table 4. Supplemental Tables 1 and 2

Correlations of morphometric data and blood analytes with month of nesting season
Complete statistical results of ANOVA and Kruskal-Wallis tests comparing morphometric data and blood analytes by month are shown in Tables 5 and 6, with graphical representation of select analytes provided in Figs. 4 and 5. Mass was the only morphometric analyte showing significant differences by month of nesting season, with an overall decline across nesting season.Blood analytes that decreased by month included PCV, amylase, calcium, cholesterol, lipase, phosphorus, triglycerides, uric acid, total protein by Biuret and refractometer, albumin, total globulins, iron, zinc, α-tocopherol, retinol and 23 fatty acids.Blood analytes that increased included BUN, chloride, potassium and sodium.Whilst not significant, β-hydroxybutyrate concentrations by month are shown in Fig. 4c.

Discussion
This study presents morphometric and blood analyte data of nesting loggerheads from a globally important nesting beach in central eastern Florida and includes routine haematology and plasma biochemistry data, as well as novel blood analytes for this life-stage class (e.g.β-hydroxybutyrate, vitamins, trace nutrients and fatty acid profiles).Specifically, BUN, βhydroxybutyrate, plasma lipids and fatty acid profiles showed utility as biomarkers of nutritional balance and provided insight into the catabolic state and physiological changes during reproductive activity and energetic demands across nesting seasons.The overall trends and correlations identified in this study revealed insights into phases of fasting and are consistent with the capital breeding strategy of nesting sea turtles as documented in loggerheads nesting on the west coast of Florida (Perrault & Stacy, 2018) and in other species, including leatherbacks (Honarvar et al., 2011;Perrault et al., 2012Perrault et al., , 2014Perrault et al., , 2016;;Plot et al., 2013;Stacy et al., 2019), green turtles (Hamann et al., 2002;Hays et al., 2002; Page-Karjian

Morphometrics
Morphometric data from loggerheads nesting at ACNWR were similar in range to a previous study from the central eastern Florida region (Bjorndal et al., 1983) and to other nesting loggerheads along the Atlantic coast of Florida and Georgia (Deem et al., 2009;Flower et al., 2015).However, mean SCL (89.9 cm) of loggerheads from this study was slightly lower than the 37-year historical mean (91.1 cm) of loggerheads nesting on ACNWR.It is notable that the minimum size of sexually mature female loggerheads and green turtles on the ACNWR was observed to have slightly reduced over the past four decades due to population-level effects that are thought to have been associated with demographic and behavioural changes and declines in habitat quality (Phillips et al., 2021)               was expected with larger turtles being heavier, a common observation in turtles of various life-stage classes (Georges & Fossette, 2006;Martins et al., 2022;Perrault et al., 2020;Stacy et al., 2023).The overall mild degree of observed injuries in this study seems representative for nesting turtles (Miller, 1997;Page-Karjian et al., 2020), although partial or even complete flipper amputations are not uncommon in sea turtles (Aoki et al., 2023;Ataman et al., 2021;Deem et al., 2006, Deem et al., 2009;Perrault et al., 2012).The degree of epifaunal fouling (including barnacles, algae and leeches) in this study was similar to findings in a large-scale study of nesting loggerheads from St. George Island, Florida, and juveniles from North Carolina (Ingels et al., 2020;Stamper et al., 2005).Leeches have also been commonly observed on the cloaca and neck of nesting loggerheads and green turtles on Juno Beach, Florida (Loggerhead Marinelife Center, unpublished data).The observed lower epibiota coverage in May compared to July and August suggests nesting turtles presumably decrease activity levels during inter-nesting intervals (Frick et al., 2000) or epibiota growth increases in warmer waters encountered adjacent to nesting beaches.

Blood analyte data
Given the lack of significant external or behavioural abnormalities, the ACNWR loggerheads can be considered clinically normal representatives of this nesting aggregation in central eastern Florida.This was also reflected in haematology, plasma biochemistry, trace nutrient and vitamin data as reference intervals were mostly within ranges reported for other nesting loggerhead populations in the USA, Canary Islands and Turkey (Deem et al., 2009;Sözbilen et al., 2018;Casal et al., 2009;Flower et al., 2015;Gicking et al., 2004;Molter et al., 2022;Perrault et al., 2016).The only analyte that was in the lower range of reported reference intervals was PCV, a haematological measure of critical clinical importance (Stacy & Innis, 2017).Compared to other studies of nesting loggerheads from the USA, PCV tended to be lower in ACNWR turtles (Deem et al., 2009;Flower et al., 2015).Considerations for this observation include differences in foraging grounds (e.g.dietary composition, abundance) preceding the nesting period, different timing of studies with variable number of study turtles, in migratory distances between foraging and nesting grounds, possibly some degree of underlying chronic but subclinical issues (e.g. from external lesions) although there was no evidence of systemic inflammation or other abnormalities in blood analytes of turtles with <20% PCV (n = 3; 15-19% during July and August) or hyporexia resulting in reduced erythropoiesis at the height of the nesting season.Although lymph contamination was not visible during collection of any blood samples, some mild degree that may  (Bolten & Bjorndal, 1992;Rousselet et al., 2013;Perrault et al., 2020).
Fatty acid profiles from blood samples have been documented in wild-captured and cold-stunned juvenile sea turtles, freshwater snapping turtles (Clyde-Brockway et al, 2021;Dass et al., 2020;Heniff et al., 2022;Koutsos et al., 2021) and for the first time in nesting sea turtles in this study.The predominant essential fatty acids in nesting turtles included monoenes, ω9 (omega-9), oleic acid 18:1ω9, saturates, palmitic acid 16:0, polyunsaturated fatty acids (PUFA), highly unsaturated fatty acids (HUFA), palmitoleic acid 16:1ω7 and ω6 (omega-6), and also the ratio of arachidonic acid to eicosapentaenoic acid (AA:EPA).Some of these are similar to lipids identified in yolk of green turtles and ostrich eggs, suggesting their involvement in vitellogenesis (Noble et al., 1996;Craven et al., 2008).These fatty acid profiles of nesting loggerheads are substantially different from profiles of foraging neritic juvenile green and Kemp's ridley sea turtles (Lepidochelys kempii), with a predominance of 18:2ω6 and 18:3ω3 in green turtles, but similar concentrations of arachidonic acid 20:4ω6, and higher EPA (20:5ω3) in Kemp's ridleys (Koutsos et al., 2021).None of these fatty acids were predominant in nesting loggerheads and were substantially lower than values for juvenile green and Kemp's ridley turtles, in which the predominant monoenes were not detected.The variable observations of plasma lipids across sea turtle studies suggest differences in diet due to species, geographical locations, season and/or life-stage classes, the need for certain circulating fatty acids necessary for vitellogenesis, various stages of fasting or hyporexia with consequent release of different components of fatty acids and components from tissues and/or changes to the primary tissues of origin (e.g.liver, adipose tissue) during phases of hyporexia (McCue, 2008;Price & Valencak, 2012;Secor & Carey, 2011).

Correlations of BCI with blood analytes
Copper and iron possibly reflect higher body stores in turtles with higher BCI.Because both elements are mostly bound to plasma proteins in circulation, their blood concentrations are largely determined by plasma protein concentrations.The decreasing trend of circulating iron across nesting season suggests utilization of retained and recycled iron stores due to fasting and/or contribution to vitellogenesis (Knutson & Wessling-Resnick, 2003).

Changes in mass and blood analytes with month of nesting
The observed differences of mass and blood analytes by nesting month confirmed expected trends associated with capital breeding in females across nesting season.Although the data herein do not reflect the same individuals across time, the effect of time appears significant in this cohort of study turtles for several blood analytes.Similar physiological changes have been documented in nesting loggerheads, leatherbacks, green turtles and hawksbills (Hamann et al., 2002;Hays et al., 2002;Perrault et al., 2012Perrault et al., , 2014Perrault et al., , 2016;;Honarvar et al., 2011;Perrault & Stacy, 2018;Goldberg et al., 2013;Page-Karjian et al., 2020).During their nesting season, sea turtles apparently forage little to none (Hopkins-Murphy et al., 2003) and concurrently have high energetic demands (e.g. for mating, nesting/crawling activities, inter-nesting movements, vitellogenesis).This is reflected in the decline in mass across month of nesting season, which has also been observed in nesting leatherbacks, green turtles and hawksbills (Hays et al., 2002;Santos et al., 2010;Goldberg et al., 2013;Plot et al., 2013).Although mass declined across nesting season, BCI tended to be lower in August without statistical significance.BCI, a calculated parameter, is considered an important indicator for overall and nutritional assessment in sea turtles (Bjorndal et al., 1983); this parameter may not similarly represent nutritional health in fasting, reproductive adult female turtles in visually adequate body condition.Thus, all available morphometric data should be considered when assessing sea turtles of various life-stage classes.
PCV tended to decrease as nesting seasons progressed, with turtles nesting in May showing the highest PCV and turtles nesting in August showing the lowest.Similar seasonal PCV reduction is seen in leatherbacks and is considered an important indicator of foraging status during nesting season, of remigration interval and potentially, of age (Perrault et al., 2016;Honarvar et al., 2011).Other indicator declines with nesting season progression were related to nutritional and energy metabolism, catabolic state and needs for vitellogenesis in ACNWR loggerheads including cholesterol, triglycerides, calcium, phosphorus, total protein (TP-R and TP-B), albumin, total globulins, iron, alpha-tocopherol, retinol and 23 of 34 fatty acids.Most of these trends were similar to leatherback turtles from Equatorial Guinea and St. Croix, US Virgin Islands and to loggerheads from Casey Key, Florida (Perrault et al., 2014(Perrault et al., , 2016;;Honarvar et al., 2011;Perrault & Stacy, 2018).The observed declines in fatty acids across nesting seasons presumptively reflect utilization of stored energy and progression of fasting state across time, whilst turtles partition energy demands towards egg production and laying.Lipid mobilization prior to and during nesting is expected, but appears to primarily rely on omega-9 fatty acids also seen as most prevalent in wild green turtles (Koutsos et al., 2021), along with use of midchain C:14 myristic acid, as well as C:16 palmitic, the most common saturated fatty acid mobilized into circulation; both are primarily utilized to anchor signalling proteins in cell membranes (Voet & Voet, 2010).by full body storage of nutrients (i.e.low BHB and BUN concentrations; high concentrations of lipids), followed by the initial phase of fasting/low food intake and use of carbohydrates (i.e. increase in BHB with a stable BUN; lipids tend to decrease due to ketogenesis) and continuing to an advanced stage of mobilization from tissues (i.e.decrease in BHB and lipids; increases in BUN as protein reserves such as muscle tissues start to be catabolized) (Cherel et al., 1988;Plot et al., 2013;Price et al., 2013;Secor & Carey, 2011).The variability in BHB associated with fasting has been documented in other reptile species; BHB decreases with longer periods of fasting (Price et al., 2013).BUN increased significantly in nesting hawksbills and, in a previous study of nesting loggerheads, had no statistically significant correlation (Perrault & Stacy, 2018;Goldberg et al., 2013).This absence of a trend was attributed to the lack of serial sampling of individual turtles throughout nesting season.In nesting leatherbacks, BUN showed an initial decline followed by an increase towards the end of nesting season (Plot et al., 2013).These variations and tendency towards a BUN increase across nesting seasons in various sea turtle species suggest a switch or beginning transition from lipid to protein catabolism at the end of nesting (Plot et al., 2013;Perrault et al., 2014, Perrault & Stacy, 2018).The degree to which these trends in nutritionally relevant blood analytes occur in individual nesting turtles may be associated with age, reproductive history (remigrant/previous nester vs. neophyte), frequencies of activities involving mating or nesting (e.g.number of clutches), dietary differences from foraging grounds or individual variation in times at which negative energy balance starts over the course of a nesting season (Goldberg et al., 2013;Perrault & Stacy, 2018;Honarvar et al., 2011).
The electrolytes sodium, chloride and potassium showed significant increases across nesting season, similar to the described trends of chloride in nesting loggerheads (Perrault & Stacy, 2018) and sodium and chloride in nesting green turtles (Page-Karjian et al., 2020;Price, 2017).Trends for sodium and potassium were opposite for nesting leatherbacks and hawksbills (Honarvar et al., 2011;Goldberg et al., 2013).These observations may suggest species-specific differences regarding haemodynamic differences from energetic expenses towards the end of nesting season, considering that vitellogenesis requires plasma proteins that also maintain the oncotic pressure within blood vessels, or alterations in salt gland metabolism.Additionally, variability in diet, water consumption and energetic expenses with migration and reproductive output may also contribute to species variations (Goldberg et al., 2013;Perrault & Stacy, 2018;Plot et al., 2013;Price et al., 2019).
The decline of α-tocopherol suggests its utilization (e.g. for vitellogenesis) and/or release from adipose tissue, respectively.The presence of γ-tocopherol in circulation is associated with increased nitrogen-based antioxidant function against inflammation; however, the concentrations seen in these sea turtles appear minimal (<1% of α-tocopherol) (Ford et al., 2006).It is also possible that plasma α-tocopherol decreases also reflect the high level of mobilization of selenium in vitellogenesis as one has a sparing effect on the other (Combs & McClung, 2017;Guirlet et al., 2008).Vitamin A, as retinol, also declined across nesting season, indicating utilization for vitellogenesis, reproduction and modulation of systemic immune health during fasting.Retinol maintains itself in circulation through liver stores, with usage generally being masked until marked deficiency.
The declines in tissue enzymes amylase and lipase by month do not seem biologically relevant as their means stayed within expected ranges for loggerheads of other life-stage classes (Casal et al., 2009;Deem et al., 2009;Kelly et al., 2015).Zinc also decreased across nesting season, possibly due to utilization or individual hormonal variations across nesting seasons, considering that individual turtles were not repeatedly sampled.

Conclusions
This study documents morphometric and blood analyte data in nesting loggerheads at ACNWR, a beach that is considered critical for the Northwest Atlantic loggerhead population (Weishampel et al., 2003;Ehrhart et al., 2014).The data herein provide insight into understanding the metabolic and energetic demands in reproductively active female sea turtles and will be useful for spatiotemporal comparisons.Further, we identified biomarkers of nutritional balance (e.g.BUN, βhydroxybutyrate, plasma lipids cholesterol and triglycerides, calcium, iron, vitamins A and E and fatty acid profiles) for assessing foraging status and phases of reproduction.These biomarkers help identify the catabolic state and thus support the hypothesis of capital breeding in nesting loggerheads.Applications for this information include assessment of individual animals in rescue or managed care settings, comparative study of local, regional, and global intra-or interspecies relationships, and assessment of informed recovery and management strategies.

Figure 1 :
Figure 1: Locations of both sampling sites of loggerhead sea turtles (Caretta caretta) at the ACNWR, Florida, USA.

Figure 2 :
Figure 2: Relationship between mass (kg) and standard straight carapace length (cm) for nesting loggerhead sea turtles (Caretta caretta) from the ACNWR.The trend was significant and the equation for the line-of-best-fit is shown with the 95% confidence interval of the regression line.

Figure 3 :
Figure 3: Relationship between BCI and plasma (a) copper and (b) iron in nesting loggerhead sea turtles (Caretta caretta) from the ACNWR.Data were log-transformed.The trends were significant and the equations for the line-of-best-fit are shown with the 95% confidence intervals of the regression line.

Figure 4 :
Figure 4: Mean ± standard deviation of mass and select blood analytes showing significant trends (with the exception of β-hydroxybutyrate, which was not significant, but tended to increase mid-season) by month of nesting season in nesting loggerhead sea turtles (Caretta caretta) from the ACNWR.Morphometric results include (A) mass, whilst included blood analytes are (B) PCV, (C) β-hydroxybutyrate, (D) blood urea nitrogen, (E) cholesterol, (F) triglycerides, (G) uric acid, (H) total protein, (I) α-tocopherol, (J) retinol and (K) sum of all detected fatty acids.ANOVA with Tukey's post hoc tests were used to compare data by month, with Kruskal-Wallis tests with Dunn's post hoc tests with Benjamini-Hochberg adjustment were used when the data did not meet the assumptions of the ANOVA.Different letters above each column represent significant differences by month at P < 0.050.

Figure 5 :
Figure 5: Mean ± standard deviation of electrolytes and minerals showing significant trends (with the exception of calcium:phosphorus, which was not significant) by month of nesting season in nesting loggerhead sea turtles (Caretta caretta) from the ACNWR.Electrolytes and minerals include (A) calcium, (B) phosphorus, (C) calcium:phosphorus, (D) chloride, (E) potassium and (F) sodium.ANOVA with Tukey's post hoc tests were used to compare data by month, with Kruskal-Wallis tests with Dunn's post hoc tests with Benjamini-Hochberg adjustment were used when the data did not meet the assumptions of the ANOVA.Different letters above each column represent significant differences by month at P < 0.050.

Table 2 :
Measures of central tendency, range and reference intervals (with 90% confidence intervals for upper and lower limits) for haematologic and plasma biochemical data in Standard International units for nesting loggerhead sea turtles (Caretta caretta) from the ACNWR.Parametric methods for sample sizes ≥20 but <120 were used to calculate reference intervals, unless otherwise indicated by footnotes.Normality was assessed using the Shapiro-Wilk test, whilst outliers were detected using the Dixon-Reed test.All plasma samples had haemolysis and lipaemia scores ≤1+, which is not considered to cause interference using dry chemistry analysis.Reference intervals could not be calculated for some variables due low sample sizes (<20) or because the majority of the values fell below the detection limit (i.e.right-skewed data)

Table 4 :
Measures of central tendency, range and reference intervals (with 90% confidence intervals for upper and lower limits) for fatty acids (in

Table 5 :
Statistical results of morphometric data and blood analytes by month of nesting season in nesting loggerhead sea turtles (Caretta caretta) from the ACNWR.Sample sizes are provided parenthetically.ANOVA with Tukey's post hoc tests were used to compare data by month, with Kruskal-Wallis (KW) tests with Dunn's post hoc tests with Benjamini-Hochberg adjustment were used when the data did not meet the assumptions of the ANOVA.The mean values are shown by month with different superscript letters indicating significant differences (at P < 0.050) as determined by post hoc tests.The test statistics are provided (F for ANOVA, H for KW), in addition to the degrees of freedom (df ), the P-value and if transformations were performed Analyte